"A PROCESS FOR THE PREPARATION OF ANTIMICROBIAL FRACTION FROM STREPTOMYCES ERUMPENS USEFUL AS THERAPEUTIC AGENT"

Abstract

The present invention relates to the process for the preparation of antimicrobial fraction, which comprises growing a novel strain Streptomyces erumpens (MTCC-3474) in a production medium, at temperature in the range of 20-45 °C, pH in the range of 4.0-10.0 for a period of 3-7 days to obtain broth, extracting the said broth with polar organic solvents and purifying the fraction by chromatographic methods. The antimicrobial fraction is useful as a therapeutic agent against a variety of gram positive and gram negative bacteria and fungi.

Full Text

The present invention relates to the process for preparation of antimicrobial fraction from Streptomyces erumpens. The present invention particularly relates to a process for the preparation of antimicrobial fraction from Streptomyces erumpens (MTCC No. 3474) useful as therapeutic agent, a novel alcohol soluble fraction.
Earlier Streptomycin erumpens Calot & Cercos (Buchnan and Gibbons, Bergys Mannual of determinative bacteriology, 8th Ed. P771; 1974) has been reported to exhibit antibacterial activity, and is inhibited by streptomycin. It produces the tetraenic antifungal antibiotic 17732 (tetrins A & B and another polyene). Poor growth was reported on czapek's agar. St. erumpens, a grey series utilises D-glucose, L-Arabinose, D-Fructose, D-Galactose, Raffinose, D-Mannitol and I-Inositol.
The discovery of streptomycin and revealing of its valuable medicinal properties were a powerful impetus in the study of streptomycete species and in the search of new strains that might produce new antibiotic substances. (Krik and Othner, Encyclopedeae of Chem.Tech. 2nd Ed. Vol. 18, 1969.)
The interest to this group of antibiotics has increased in recent years. At the present time a great number of antibiotics (over 6000) produced by bacteria, fungi, algae, higher plants and animals have been described. But most important of them are mainly produced by Streptomycetes species (S.A. Waksman and H.A Leehevalier, The actinomycetes Vol III, Bailliere, Tinall & Cox, Ltd, London 1962). This type of compounds are isolated from fermentation broth of various Streptomyces species are highly oxygenated derivatives and they differ only by variation of a few substitutents of the naphthacene skeleton. (Janos Berdy, Hand book of antibiotic compounds volume III ACRC Press P.37 1978). The antibiotic properties of Streptomyces sp. and their occurrence in nature are being studied at the present time to enrich science and practical medicine by new very interesting antibiotics that are used in various branches of medicine.

The main object of the present invention is to provide a process for the preparation of antimicrobial fraction from Streptomyces erumpens, (MTCC No. 3474) useful as therapeutic agent. To isolate novel antimicrobial compounds from microbes, wherein we have screened many organisms from different soil samples of terrestrial and marine sources and isolated a new strain designated as Streptomyces erumpens (MTCC No. 3474) useful as therapeutic agent to produce potent antimicrobial compound. The new strain (MTCC No. 3474) useful as therapeutic agent is easy to grow and maintain, and multiplies profusely on starch, casein agar medium in about 4-7 days. The production of antibiotic is good in a conventional medium at optimum pH and optimum temperature.
The present strain Streptomyces erumpens is an actinomycete, gram positive, filamentous form with spiral spore chains. The temperature range for the growth is 25°C to 45°C and the pH range is 5.0 to 10.0. The sodium chloride tolerance is from 2.5 to 4.0%. Table 1 shows colony characters, Table 2 shows physiological characteristics and Table 3 shows biochemical properties. Table 4 shows acid production and Tween hydrolysis. Table 5 shows utilization of carbohydrates. Table 6 shows growth characters on different growth media.
Based on the above properties the strain is identified as Streptomyces erumpens (MTCC NO. 3474). It differs from the type strain Streptomyces erumpens (Calot & Cercos) in the following properties. Optimum pH is 6.0-8.5, Optimum temperature 20°C - 40°C, NaCl tolerence 2.5%-4.9%. There is no growth at 55°C. The present strain cannot tolerate >5% NaCl concentration. It cannot grow on raffinose and meso inositol where as the type strain St.erumpens grows on raffinose and I-inositol. The present strain produces novel antibiotic fraction under optimum temperature, pH and growth conditions. The growth conditions are requirement of carbon and nitrogen sources along with macro and micro-nutrients for optimum yield of antimicrobial fraction.
Another object of the present invention is the isolation, separation and partial purification of bioactive antimicrobial fraction for the first time from a new strain of Streptomyces erumpens (MTCC No. 3474) for therapeutic uses. The bioassay on variety of microorganisms shows that it is active on both gram positive and gram negative bacteria and fungi.

SM— Substrate mycelium
AM—Aerial mycelium
ISP—International Streptomycetes Project
Still another object of the present invention is that the new source for a new antimicrobial fraction, which is showing very good broad spectrum antimicrobial activity may be used as a new antibiotic with simple isolation process and might result in a new potent source of pharmaceutielly active drug in near future by structure identification and modification, by formulating the same fraction as such and/or increasing the yield of this fraction by genetic manipulation of the organism St.

erumpens (MTCC NO. 3474) strain for therapeutic uses. This organism is very stable and there is no loss of activity upon sub-culturing.
Accordingly the present invention provides a process for preparation of antimicrobial fraction as a therapeutic agent, which comprises growing a novel strain Streptomyces erumpens (MTCC No.3474) in a production medium such as herein described, at temperature in the range of 20-45 °C, pH in the range of 4.0-10.0 for a period of 3-7 days to obtain broth, extracting the said broth with polar organic solvents and purifying the fraction by chromatographic methods.
In an embodiment of the present invention the production medium used is selected from nutrient mineral salts medium and is supplemented with protein content source selected from corn steep liquor, soyabean meal, casein, malt extract and yeast extract.
In another embodiment of the present invention the organic solvents used for extraction are selected from ethylacetate, butanol, methanol, ethanol and mixture thereof.
In yet another embodiment of the present invention the chromatographic method used is selected from thin layer chromatography, column chromatography, high-pressure liquid chromatography.
The process for the preparation of a novel antimicrobial fraction from Streptomyces erumpens (MTCC no.3474), a strain to produce potent antimicrobial compound, isolated in our pursuit to identify novel antimicrobial compounds from different soil samples. The new strain is identified based on morsphological, physiological and biochemical characteristics described in table as Streptomyces erumpens (MTCC NO. 3474), which was grown in carbon and nitrogen supplimented broth at optimum pH and temperature and extracting the said actimomycete culture broth with ethylacetate in combination with methanol, ethanol to extract thoroughly the antimicrobial fraction in a known manner

to get crude fraction and partially purifying the antimicrobial fraction by conventional chromatography to recover the active fraction.
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.
Example-1
5 day old culture borth (2 Lt) of Streptomyces erumpens is prepared with corn steep
liquor supplemented medium consisting of (g/Lt) Corn steep liquor 10, Soluble starch
15. Dextrose 15. CaCO3 7, CaCl2 0.0001 at optimum pH and temperature and
sterilized at 15 pressure for 20 mts. After it is cooled to room temperature 10%
inoculum grown for 24 hr. in growth medium is transferred aseptically to the above
production medium and kept on shaker at 150 RPM for 5 days at 28 + 2°C is extracted
thoroughly with 1 litre of 8:2 ethyl acetate and methanol. The active organic crude
extract (1100 mg) is used for further purification. The active compound purified by
ascending paper chromatrography using 4% acetic acid Butanol saturated with water
as mobile phase. The active band is eluted with methanol to get colourless viscous
antimicrobial fraction (yield 39.14 mg)
Example-2
6 day old culture broth (3 lt) of Streptomyces erumpens grown in corn steep liquor
amended medium kept on orbital shaker at 28 + 2°C at 180 RPM is extracted
thoroughly with 1.5 lt of 7:3 ethylacetate and methanol (1650 mg). The organic crude
extracts pooled evarporated under vaccum at 40°C is purified by chromatography
using acetic acid, butanol saturated with water and eluted with methanol to get active
fraction (yield 71.9 mg)
Example-3

7 day old culture broth (4 It) of St. erumpens grown in corn steep liquor amended medium at 30 + 2°C at 170 RPM is extracted thoroughly with 6:4 ethylacetate methanol. The organic extracts pooled evaporated under vaccum at 38°C to get (2198 mg) of crude fraction, which is purified by chromotography using butanol acetic acid saturated with water as mobile phase. The active band is eiuted with methanol to get active colourless viscous antimicrobial compound (yield 83.45)
Example-4
5 day old culture broth 2.5 It of St. erumpens grown in amended medium at 28°C at 160 RPM is extracted thoroughly with 6.5 : 3.5 ethylacetate, methanol. The organic extracts pooled,,evaporated under vaccum at 37°C to get 1402 mg crude fraction, which is purified by chromatography using butanol saturated with water and 3.5% acetic acid as mobile phase. The active band is eiuted with methanol to get active antimicrobial fraction (yield 59.982 mg).
Example-5
4 day old culture broth 3.5 It of Streptomyces erumpens grown in corn steep amended medium is extracted throughly with ethylacetate in combination with methanol. The organic extracts pooled, evaporated under vaccum at 36°C to get 1898.5mg of crude fraction which is purified by chromatography using butanol saturated with water as mobile phase. The active band is eiuted with methanol to get active fraction (yield 71.82 mg)
Anti microbial Assay:
All the media used are from Hi Media, Bombay, India. Bacterial strains were grown on nutrient agar and suspended in Mueller Hinton broth and fungal strains in sabouroud broth. A conventional two fold serial dilution method is employed to determine minimum inhibitory concentration (Jones et al, 1984;In: Lenette EH, Ballows et al., Manual of Clinical Microbiol.,972-977, Washington DC, American Society for Microbiol).The compound disso n acetone at 2mg/ml concentration is diluted in respective broths in the range of l00-1 .56µg/ml. Cultures grown at 37°C for 20 h were used as inoculum(approximately l05-6 CFU/ml). Test cultures were

incubated at 37° C for 24h. All the results (average of triplicates) were presented in µg/ml (Table 5). The lowest concentration of antimicrobial agent that results in the complete inhibition of microorganism represents the minimum inhibitory concentration (MIC (ng/ml).
The activity against the following organisms have been tested and the minimum inhibitory concentration against test microbes is presented in Table 7.
Table 7: Antimicrobial Activity of Streptomyces erumpens (MTCC NO. 3474) fraction.

(Table Removed)

We Claim:
1. A process for preparation of antimicrobial fraction as a therapeutic agent, which comprises growing a novel strain Streptomyces erumpens (MTCC No.3474) in a production medium such as herein described, at temperature in the range of 20-45 °C, pH in the range of 4.0-10.0 for a period of 3-7 days to obtain broth, extracting the said broth with polar organic solvents and purifying the fraction by chromatographic methods.
2. A process as claimed in claim 1, wherein the production medium used is selected from nutrient mineral salts medium and is supplemented with protein content source selected from corn steep liquor, soyabean meal, casein, malt extract and yeast extract.
3. A process as claimed in claim 1-2 wherein the organic solvents used for extraction are selected from ethylacetate, butanol, methanol, ethanol and mixture thereof.
4. A process as claimed in claims 1-3 wherein the chromatographic method used is selected from thin layer chromatography, column chromatography, high-pressure liquid chromatography.
5. A process for the preparation of antimicrobial fraction such as herein described in the specification with reference to examples.